Dig dna labelling and detection kit

Site-directed mutagenesis on the ureA::gfp gene was performed by the fusion PCR technique [79 (link)] using KAPA HiFi DNA polymerase (KAPA Biosystems) with primers Ure5-F, Ure3-R and complementary ones carrying the desired substitution (see the electronic supplementary material, table S3). DNA extracted from a ureA::gfp::AFpyrG strain (MVD 10A) was used as a template. A resulting 7 kb fusion product was amplified with nested primers Ure5-N and Ure3-N and purified with the GeneJET Gel Extraction Kit (Thermo Scientific). The resulting construct was transformed in a ureAΔ::riboB pyrG89 pyroA4 riboB2 nkuAΔ::argB veA1 strains (MVD 13A and isogenic yA2 MVD 14A). Protoplasts were plated on selective (lacking uridine and uracil) 1 M sucrose MM and incubated at 37°C. Transformants were purified on selective MM, and its ability to grow in different concentrations of urea and 2-thiorea was tested. Transformants showing a riboB2 phenotype were preferentially selected for further analysis as this was indicative of integration of the construct at the ureA locus. Assessment of single copy or multicopy transformants was performed by a DIG DNA labelling and detection kit (Roche Applied Science). Sequencing of the resulting mutants was performed in Macrogen Inc. (Seoul, Korea)

Site-directed mutagenesis on the ureA::gfp gene was performed by the Fusion-PCR technique [55 (link)] using KAPA HiFi DNA polymerase (KAPA Biosystems) with primers Ure5-F, Ure3-R and complementary ones carrying the desired substitution (electronic supplementary material, table S2). DNA extracted from a ureA::gfp::AFpyrG strain (MVD 10A) was used as template. A resulting 7 kb fusion product was amplified with nested primers Ure5-N and Ure3-N and purified with the GeneJET Gel Extraction Kit (Thermo Scientific). The resulting construct was transformed in a ureAΔ::riboB pyrG89 pyroA4 riboB2 nkuAΔ::argB veA1 strain (MVD 13A). Protoplasts were plated on selective (containing riboflavin, but not uridine and uracil) 1 M sucrose MM and incubated at 37°C. Transformants were purified on selective MM and its ability to grow both in different concentrations of urea and 2-thiourea was tested. Southern blots were performed in order to check the integration to the ureA locus and to assess the number of construction copies integrated in the genome. Probes were labelled with the DIG DNA labelling and detection kit (Roche Applied Science). Sequencing of the resulting mutants was performed at Macrogen, Inc. (Seoul, Korea).

Total small RNA population was isolated from the leaves of transgenic and NT control plants^{43 (link)}. It was performed to assess the expression level of siRNA in transgenic (T₀, T₁) plants with respect to corresponding non-transformed cotton plants. The low molecular weight RNA were separated on denaturing polyacrylamide gel (PAGE 15%) and transferred onto a Hybond-N nylon membrane (GE Healthcare, UK). Hybridization was performed with CLCuMuV-C4 gene-based DIG-11-dUTP labelled probe at 42 °C in DIG Easy Hyb solution using DIG DNA labelling and detection kit (Roche Diagnostics, Germany). The membrane was equilibrated in Detection buffer containing NBT/BCIP-T under Gel Doc™ EZ System (BioRad, USA) using the Image Lab™ software Version 4.1. siRNA fragments (20–25 nt long) were visualized, which were compared with the original PAGE exhibiting ultra-low range RNA marker (Thermo Scientific, Germany).